Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability
Jayne M. Squirrell1, David L. Wokosin2, John G. White2
& Barry D. Bavister1
1
Animal Health and Biomedical Sciences, University of Wisconsin−Madison, Madison, WI 53706
2
Integrated Microscopy Resource, University of Wisconsin−Madison, Madison, WI 53706
Correspondence should be addressed to Jayne M. Squirrell jsquirre@students.wisc.edutwo-photon microscopylaser scanning confocal microscopylive cell fluorescence imagingembryomitochondrial dynamicsmammalhamster
A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H2O2 may account, in part, for this inhibition. Thus, two-photon microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.
